Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters.

The high-affinity/low-capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues and organs. Information regarding PepT2 in teleost fish is limited and, to date, functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and the distal part of the gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp recordings of transport and presteady-state currents. Both PepT2a and PepT2b in the presence of Gly-Gln elicit pH-dependent and Na+ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high-affinity/low-capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2-type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es). KEY POINTS: Two slc15a2-type genes, slc15a2a and slc15a2b coding for PepT2-type peptide transporters were found in the Atlantic salmon. slc15a2a transcripts, widely distributed in the fish tissues, are abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and distal gastrointestinal tract. Amino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. Detailed kinetic analysis indicates that both PepT2a and PepT2b operate as high-affinity transporters. The kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters.

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure-Function Relationships in Mammalian SLC6 Transporters.

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional-structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

Identification of SLC15A4/PHT1 Gene Products Upregulation Marking the Intestinal Epithelial Monolayer of Ulcerative Colitis Patients.

SLC15A4/PHT1 is an endolysosome-resident carrier of oligopeptides and histidine recently come into view as a key path marker of immune/autoimmune/inflammatory pathways in immune cells. Yet, its emerging role in inflammatory processes directly targeting the gastrointestinal epithelial layer, as in the multifactorial pathophysiology of inflammatory bowel disease (IBD), is poorly investigated. Here, the first identification of SLC15A4/PHT1 gene products in human colonic epithelium of ulcerative colitis (UC) patients is reported, showing protein primarily localized in intracellular vesicle-like compartments. Qualitative and quantitative immunohistochemical analyses of colon biopsies revealed overexpression of SLC15A4/PHT1 protein product in the epithelial layer of UC patients. Results were successfully mirrored in vitro, in spontaneously differentiated enterocyte-like monolayers of Caco-2 cells specifically exposed to DSS (dextran sodium sulphate) to mimic IBD inflammatory onsets. SLC15A4/PHT1 expression and cellular localization were characterized confirming its (dys)regulation traits in inflamed vs. healthy epithelia, strongly hinting the hypothesis of SLC15A4/PHT1 increased function associated with epithelial inflammation in IBD patients.

Morpho-functional remodelling of the adult zebrafish (Danio rerio) heart in response to waterborne angiotensin II exposure.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System, is a pluripotent humoral agent whose biological actions include short-term modulations and long-term adaptations. In fish, short-term cardio-tropic effects of AngII are documented, but information on the role of AngII in long-term cardiac remodelling is not fully understood. Here, we describe a direct approach to disclose long-term morpho-functional effects of AngII on the zebrafish heart. Adult fish exposed to waterborne teleost analogue AngII for 8 weeks showed enhanced heart weight and cardio-somatic index, coupled to myocardial structural changes (i.e. augmented compacta thickness and fibrosis), and increased heart rate. These findings were paralleled by an up-regulation of type-1 and type-2 AngII receptors expression, and by changes in the expression of GATA binding protein 4, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and superoxide dismutase 1 soluble mRNAs, as well as of cytochrome b-245 beta polypeptide protein, indicative of cardiac remodelling. Our results suggest that waterborne AngII can sustain and robustly affect the cardiac morpho-functional remodelling of adult zebrafish.

Identification and characterization of the Atlantic salmon peptide transporter 1a.

Peptide transporter 1 (PepT1) mediates the uptake of dietary di-/tripeptides in vertebrates. However, in teleost fish gut, more than one PepT1-type transporter might operate, because of teleost-specific whole gen(om)e duplication event(s) that occurred during evolution. Here, we describe a novel teleost di-/tripeptide transporter, i.e., the Atlantic salmon (Salmo salar) peptide transporter 1a [PepT1a; or solute carrier family 15 member 1a (Slc15a1a)], which is a paralog (77% similarity and 64% identity at the amino acid level) of the well-described Atlantic salmon peptide transporter 1b [PepT1b, alias PepT1; or solute carrier family 15 member 1b (Slc15a1b)]. Comparative analysis and evolutionary relationships of gene/protein sequences were conducted after ad hoc database mining. Tissue mRNA expression analysis was performed by quantitative real-time PCR, whereas transport function analysis was accomplished by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp measurements. Atlantic salmon pept1a is highly expressed in the proximal intestine (pyloric ceca ≈ anterior midgut > midgut >> posterior midgut), in the same gut regions as pept1b but notably ~5-fold less abundant. Like PepT1b, Atlantic salmon PepT1a is a low-affinity/high-capacity system. Functional analysis showed electrogenic, Na+-independent/pH-dependent transport and apparent substrate affinity (K0.5) values for Gly-Gln of 1.593 mmol/L at pH 7.6 and 0.076 mmol/L at pH 6.5. In summary, we show that a piscine PepT1a-type transporter is functional. Defining the role of Atlantic salmon PepT1a in the gut will help to understand the evolutionary and functional relationships among peptide transporters. Its functional characterization will contribute to elucidate the relevance of peptide transporters in Atlantic salmon nutritional physiology.

Carnosine modulates the Sp1-Slc31a1/Ctr1 copper-sensing system and influences copper homeostasis in murine CNS-derived cells.

Carnosine (CAR) is an endogenous dipeptide physiologically present in excitable tissues, such as central nervous system (CNS) and muscle. CAR is acknowledged as a substrate involved in many homeostatic pathways and mechanisms and, due to its biochemical properties, as a molecule intertwined with the homeostasis of heavy metals such as copper (Cu). In CNS, Cu excess and dysregulation imply oxidative stress, free-radical production, and functional impairment leading to neurodegeneration. Here, we report that CAR intercepts the regulatory routes of Cu homeostasis in nervous cells and tissues. Specifically, in a murine neuron-derived cell model, i.e., the B104 neuroblastoma cells, extracellular CAR exposure up to 24 h influenced intracellular Cu entry and affected (downregulated) the key Cu-sensing system, consisting of the gene coding for the Slc31a1 transmembrane Cu importer (alias Ctr1), and the gene coding for the Cu-responsive transcription factor Sp1 ( Sp1). Also, CAR exposure upregulated CAR biosynthesis ( Carns1), extracellular degradation ( Cndp1), and transport ( Slc15a4, alias Pht1) genes and elicited CAR intracellular accumulation, contributing to the outline of functional association between CAR and Cu within the cell. Interestingly, the same gene modulation scheme acting in vitro operates in vivo in brains of mice undergoing dietary administration of CAR in drinking water for 2 wk. Overall, our findings describe for the first time a regulatory interaction between CAR and Cu pathways in CNS and indicate CAR as a novel active molecule within the network of ligands and chaperones that physiologically regulate Cu homeostasis.

Fishing in the Cell Powerhouse: Zebrafish as A Tool for Exploration of Mitochondrial Defects Affecting the Nervous System.

The zebrafish (Danio rerio) is a small vertebrate ideally suited to the modeling of human diseases. Large numbers of genetic alterations have now been modeled and could be used to study organ development by means of a genetic approach. To date, limited attention has been paid to the possible use of the zebrafish toolbox in studying human mitochondrial disorders affecting the nervous system. Here, we review the pertinent scientific literature discussing the use of zebrafish in modeling gene mutations involved in mitochondria-related neurological human diseases. A critical analysis of the literature suggests that the zebrafish not only lends itself to exploration of the pathological consequences of mitochondrial energy output on the nervous system but could also serve as an attractive platform for future drugs in an as yet untreatable category of human disorders.

Responsiveness of Carnosine Homeostasis Genes in the Pancreas and Brain of Streptozotocin-Treated Mice Exposed to Dietary Carnosine.

In excitable tissues, the endogenous dipeptide carnosine (CAR, ß-Ala-l-His) sustains homeostatic responses to various challenges. By eliciting hypoglycemic effects via actions on the autonomic nervous system and protection of pancreatic beta-cells, CAR is also relevant in diabetes. We investigated the expression of genes involved in CAR biosynthesis, degradation, and membrane transport pathways, in the pancreas and brains of mice treated with streptozotocin (STZ) and then exposed to dietary CAR. We induced hyperglycemia by STZ intraperitoneal injections; then, STZ-treated mice received drinking water with or without CAR for two weeks. We report that CAR administration elicits beneficial effects on blood glucose levels and weight loss in STZ-treated mice and, remarkably, on the insulin gene products in the pancreas, preserving gene expression from STZ challenge. Also, we describe mRNA downregulation of the Slc15a2/Pept2 (dipeptide transporter) and Cndp2 (intracellular dipeptidase) genes in the pancreas of hyperglycemic mice, and dysregulation of Carns1 (CAR synthase), Pept2 and Cndp2 in brains; interestingly, dietary CAR elicits counteracting effects. These expression patterns associate with variations of CAR content in tissues of mice. Overall, our report suggests a direct role of CAR in the diabetes-affected pancreas and in the diabetes-targeted CNS, proposing (dys)regulation of CAR's homeostasis as a marker condition.

Molecular and expression analysis of the Allograft inflammatory factor 1 (AIF-1) in the coelomocytes of the common sea urchin Paracentrotus lividus.

Allograft inflammatory factor 1 (AIF-1) is a highly conserved gene involved in inflammation, cloned and characterized in several evolutionary distant animal species. Here, we report the molecular identification, characterization and expression of AIF-1 from the common sea urchin Paracentrotus lividus. In this species, AIF-1 encodes a predicted 151 amino acid protein with high similarity to vertebrate AIF-1 proteins. Immunocytochemical analyses on coelomocytes reveal localization of the AIF-1 protein in amoebocytes (perinuclear cytoplasmic zone) and red sphaerulocytes (inside granules), but not in vibratile cells and colorless sphaerula cells. The significant increase of AIF-1 expression (mRNA and protein) found in the coelomocytes of the sea urchin after Gram + bacterial challenge suggests the involvement of AIF-1 in the inflammatory response. Our analysis on P. lividus AIF-1 contributes to elucidate AIF-1 function along the evolutionary scale and consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths.

Teleost fish models in membrane transport research: the PEPT1(SLC15A1) H+-oligopeptide transporter as a case study.

Human genes for passive, ion-coupled transporters and exchangers are included in the so-called solute carrier (SLC) gene series, to date consisting of 52 families and 398 genes. Teleost fish genes for SLC proteins have also been described in the last two decades, and catalogued in preliminary SLC-like form in 50 families and at least 338 genes after systematic GenBank database mining (December 2010-March 2011). When the kinetic properties of the expressed proteins are studied in detail, teleost fish SLC transporters always reveal extraordinary 'molecular diversity' with respect to the mammalian counterparts, which reflects peculiar adaptation of the protein to the physiology of the species and/or to the environment where the species lives. In the case of the H+ -oligopeptide transporter PEPT1(SLC15A1), comparative analysis of diverse teleost fish orthologs has shown that the protein may exhibit very eccentric properties in terms of pH dependence (e.g., the adaptation of zebrafish PEPT1 to alkaline pH), temperature dependence (e.g., the adaptation of icefish PEPT1 to sub-zero temperatures) and/or substrate specificity (e.g., the species-specificity of PEPT1 for the uptake of l-lysine-containing peptides). The revelation of such peculiarities is providing new contributions to the discussion on PEPT1 in both basic (e.g., molecular structure-function analyses) and applied research (e.g., optimizing diets to enhance growth of commercially valuable fish).

Electrodeposition of nanostructured bioactive hydroxyapatite-heparin composite coatings on titanium for dental implant applications.

In this paper we describe the one-pot fabrication of hydroxyapatite (HA)-heparin composites by electrodeposition onto Ti substrates and their characterisation in terms of structure, morphology, heparin content and bioactivity. HA coatings are well known and widely applied osteointegration enhancers, but post-implant healing rate in dental applications is still suboptimal: e.g. coagulation control plays a key role and the incorporation of an anticoagulant is considered a highly desirable option. In this study, we have developed an improved, simple and robust growth procedure for single-phase, pure HA-heparin films of thickness 1/3 µm. HA-heparin, forming nanowires, has the ideal morphology for bone mineralisation. Staining assays revealed homogeneous incorporation of sizable amounts of heparin in the composite films. The bioactivities of the HA and HA-heparin coatings on Ti were compared by HeLa cell proliferation/viability tests and found to be enhanced by the presence of the anticoagulant.

Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites.

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurological disease with mutations in SACS, encoding sacsin, a multidomain protein of 4,579 amino acids. The large size of SACS and its translated protein has hindered biochemical analysis of ARSACS, and how mutant sacsins lead to disease remains largely unknown. Three repeated sequences, called sacsin repeating region (SRR) supradomains, have been recognized, which contribute to sacsin chaperone-like activity. We found that the three SRRs are much larger (≥1,100 residues) than previously described, and organized in discrete subrepeats. We named the large repeated regions Sacsin Internal RePeaTs (SIRPT1, SIRPT2, and SIRPT3) and the subrepeats sr1, sr2, sr3, and srX. Comparative analysis of vertebrate sacsins in combination with fine positional mapping of a set of human mutations revealed that sr1, sr2, sr3, and srX are functional. Notably, the position of the pathogenic mutations in sr1, sr2, sr3, and srX appeared to be related to the severity of the clinical phenotype, as assessed by defining a severity scoring system. Our results suggest that the relative position of mutations in subrepeats will variably influence sacsin dysfunction. The characterization of the specific role of each repeated region will help in developing a comprehensive and integrated pathophysiological model of function for sacsin.

Development of L-Lysine-Loaded PLGA Microparticles as a Controlled Release System for Angiogenesis Enhancement.

Vascularization is a highly conserved and considerably complex and precise process that is finely driven by endogenous regulatory processes at the tissue and systemic levels. However, it can reveal itself to be slow and inadequate for tissue repair and regeneration consequent to severe lesions/damages. Several biomaterial-based strategies were developed to support and enhance vasculogenesis by supplying pro-angiogenic agents. Several approaches were adopted to develop effective drug delivery systems for the controlled release of a huge variety of compounds. In this work, a microparticulate system was chosen to be loaded with the essential amino acid L-lysine, a molecule that has recently gained interest due to its involvement in pro-angiogenic, pro-regenerative, and anti-inflammatory mechanisms. Poly (lactic-co-glycolic acid), the most widely used FDA-approved biodegradable synthetic polymer for the development of drug delivery systems, was chosen due to its versatility and ability to promote neovascularization and wound healing. This study dealt with the development and the effectiveness evaluation of a PLGA-based microparticulate system for the controlled release of L-lysine. Therefore, in order to maximize L-lysine encapsulation efficiency and tune its release kinetics, the microparticle synthesis protocol was optimized by varying some processing parameters. All developed formulations were characterized from a morphological and physicochemical point of view. The optimized formulation was further characterized via the evaluation of its preliminary biological efficacy in vitro. The cellular and molecular studies revealed that the L-lysine-loaded PLGA microparticles were non-toxic, biocompatible, and supported cell proliferation and angiogenesis well by stimulating the expression of pro-angiogenic genes such as metalloproteinase-9, focal adhesion kinases, and different growth factors. Thus, this work showed the potential of delivering L-lysine encapsulated in PLGA microparticles as a cost-effective promoter system for angiogenesis enhancement and rapid healing.

TEER and Ion Selective Transwell-Integrated Sensors System for Caco-2 Cell Model.

Monitoring of ions in real-time directly in cell culture systems and in organ-on-a-chip platforms represents a significant investigation tool to understand ion regulation and distribution in the body and ions' involvement in biological mechanisms and specific pathologies. Innovative flexible sensors coupling electrochemical stripping analysis (square wave anodic stripping voltammetry, SWASV) with an ion selective membrane (ISM) were developed and integrated in Transwell™ cell culture systems to investigate the transport of zinc and copper ions across a human intestinal Caco-2 cell monolayer. The fabricated ion-selective sensors demonstrated good sensitivity (1 × 10-11 M ion concentration) and low detection limits, consistent with pathophysiological cellular concentration ranges. A non-invasive electrochemical impedance spectroscopy (EIS) analysis, in situ, across a selected spectrum of frequencies (10-105 Hz), and an equivalent circuit fitting were employed to obtain useful electrical parameters for cellular barrier integrity monitoring. Transepithelial electrical resistance (TEER) data and immunofluorescent images were used to validate the intestinal epithelial integrity and the permeability enhancer effect of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) treatment. The proposed devices represent a real prospective tool for monitoring cellular and molecular events and for studies on gut metabolism/permeability. They will enable a rapid integration of these sensors into gut-on-chip systems.

Shaping the cardiac response to hypoxia: NO and its partners in teleost fish.

The reduced availability of dissolved oxygen is a common stressor in aquatic habitats that affects the ability of the heart to ensure tissue oxygen supply. Among key signalling molecules activated during cardiac hypoxic stress, nitric oxide (NO) has emerged as a central player involved in the related adaptive responses. Here, we outline the role of the nitrergic control in modulating tolerance and adaptation of teleost heart to hypoxia, as well as major molecular players that participate in the complex NO network. The purpose is to provide a framework in which to depict how the heart deals with limitations in oxygen supply. In this perspective, defining the relational interplay between the multiple (sets of) proteins that, due to the gene duplication events that occurred during the teleost fish evolutive radiation, do operate in parallel with similar functions in the (different) heart (districts) and other body districts under low levels of oxygen supply, represents a next goal of the comparative research in teleost fish cardiac physiology.

An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System.

Alamandine is a peptide of the Renin Angiotensin System (RAS), either generated from Angiotensin A via the Angiotensin Converting Enzyme 2 (ACE2), or directly from Ang-(1-7). In mammals, it elicits cardioprotection via Mas-related G-protein-coupled receptor D (MrgD), and the NOS/NO system. In teleost fish, RAS is known to modulate heart performance. However, no information is available on the presence of a cardioactive ACE2/Alamandine axis. To fill this gap, we used the cyprinid teleost Carassius auratus (goldfish) for in silico and in vitro analyses. Via the NCBI Blast P suite we found that in cyprinids ace2 is phylogenetically detectable in a subcluster of proteins including ace2-like isoforms, and is correlated with a hypoxia-dependent pathway. By real-time PCR, Western Blotting, and HPLC, ACE2 and Alamandine were identified in goldfish heart and plasma, respectively. Both increased after chronic exposure to low O2 (2.6 mg O2 L-1). By using an ex-vivo working goldfish-heart preparation, we observed that in vitro administration of exogenous Alamandine dose-dependently stimulates myocardial contractility starting from 10-11 M. The effect that involved Mas-related receptors and PKA occurred via the NOS/NO system. This was shown by exposing the perfused heart to the NOS inhibitor L-NMMA (10-5 M) that abolished the cardiac effect of Alamandine and was supported by the increased expression of the phosphorylated NOS enzyme in the extract from goldfish heart exposed to 10-10 M Alamandine. Our data are the first to show that an ACE2/Alamandine axis is present in the goldfish C. auratus and, to elicit cardiac modulation, requires the obligatory involvement of the NOS/NO system.

Grape Pomace Extract Attenuates Inflammatory Response in Intestinal Epithelial and Endothelial Cells: Potential Health-Promoting Properties in Bowel Inflammation.

Inflammatory bowel disease (IBD) implies the chronic inflammation of the gastrointestinal tract, combined with systemic vascular manifestations. In IBD, the incidence of cardiovascular disease appears to be related to an increase of oxidative stress and endothelial dysfunction. Grape pomace contains high levels of anti-oxidant polyphenols that are able to counteract chronic inflammatory symptoms. The aim of this study was to determine whether grape pomace polyphenolic extract (GPE) was able to mitigate the overwhelming inflammatory response in enterocyte-like cells and to improve vascular function. Intestinal epithelial Caco-2 cells, grown in monolayers or in co-culture with endothelial cells (Caco-2/HMEC-1), were treated with different concentrations of GPE (1, 5, 10 µg/mL gallic acid equivalents) for 2 h and then stimulated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α for 16 h. Through multiple assays, the expression of intestinal and endothelial inflammatory mediators, intracellular reactive oxygen species (ROS) levels and NF-κB activation, as well as endothelial-leukocyte adhesion, were evaluated. The results showed that GPE supplementation prevented, in a concentration-dependent manner, the intestinal expression and release of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinases (MMP)-9 and MMP-2. In Caco-2 cells, GPE also suppressed the gene expression of several pro-inflammatory markers, such as IL-1ß, TNF-α, macrophage colony-stimulating factor (M-CSF), C-X-C motif ligand (CXCL)-10, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (COX)-2. The GPE anti-inflammatory effect was mediated by the inhibition of NF-κB activity and reduced intracellular ROS levels. Furthermore, transepithelial GPE suppressed the endothelial expression of IL-6, MCP-1, VCAM-1, and ICAM-1 and the subsequent adhesion of leukocytes to the endothelial cells under pro-inflammatory conditions. In conclusion, our findings suggest grape pomace as a natural source of polyphenols with multiple health-promoting properties that could contribute to the mitigation of gut chronic inflammatory diseases and improve vascular endothelial function.

First evidence for N7-Platinated Guanosine derivatives cell uptake mediated by plasma membrane transport processes.

Nucleos(t)ide analogues (NA) belong to a family of compounds widely used in anticancer/antiviral treatments. They generally exhibit a cell toxicity limited by cellular uptake levels and the resulting nucleos(t)ides metabolism modifications, interfering with the cell machinery for nucleic acids synthesis. We previously synthesized purine nucleos(t)ide analogues N7-coordinated to a platinum centre with unaltered sugar moieties of the type: [Pt(dien)(N7-dGuo)]2+ (1; dien = diethylenetriamine; dGuo = 2'-deoxy-guanosine), [Pt(dien)(N7-dGMP)] (2; dGMP = 5'-(2'-deoxy)-guanosine monophosphate), and [Pt(dien)(N7-dGTP)]2- (3; dGTP = 5'-(2'-deoxy)-guanosine triphosphate), where the indicated electric charge is calculated at physiological pH (7.4). In this work, we specifically investigated the uptake of these complexes (1-3) at the plasma membrane level. Specific experiments on HeLa cervical cancer cells indicated a relevant cellular uptake of the model platinated deoxynucleos(t)ide 1 and 3 while complex 2 appeared unable to cross the cell plasma membrane. Obtained data buttress an uptake mechanism involving Na+-dependent concentrative transporters localized at the plasma membrane level. Consistently, 1 and 3 showed higher cytotoxicity with respect to complex 2 also suggesting selective possible applications as antiviral/antitumor drugs among the used model compounds.

Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine.

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer's integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.